Many problems associated with antisera were circumvented with the seminal discovery of mouse hybridomas capable of secreting specific monoclonal antibodies (MAbs) against predefined antigens by Kohler and Milstein (Kohler G. and Milstein C., 1975 Nature 256: 495). Since the report of Kohler and Milstein, the production of mouse monoclonal antibodies has become routine.
Monoclonal antibodies are produced by hybrid cells that result from a fusion between normal B-lymphocytes and myeloma cells. The myeloma cell lines used for fusion are B-lymphocyte tumor cell lines that grow well in vitro and can propagate indefinitely, in contrast to normal B-lymphocytes that cannot replicate or produce antibody in vitro for more than a few days. Cells derived from a fusion of the two types of cells combine the in vitro growth characteristics of the myeloma cell line with the production of an antibody derived from the B-lymphocyte.
Hybrid cells (hybridomas) are generally produced from mass fusions between murine splenocytes, which are highly enriched for B-lymphocytes, and myeloma “fusion partner cells” (B. Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994); E. Harlow et al., Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The cells in the fusion are subsequently distributed into pools that can be analyzed for the production of antibodies with the desired specificity. Pools that test positive can be further subdivided until single cell clones are identified that produce antibodies of the desired specificity. Antibodies produced by such clones are called monoclonal antibodies.
Many investigators have attempted to generate human monoclonal antibodies by generating hybridomas with human B-lymphocytes (N. Chiorazzi et al, J. Exp. Med. 156:930 (1982); C. M. Croce et al., Nature 288:488 (1980); P. A. Edwards et al, Eur. J. Immunol. 12:641 (1982); R. Nowinski et al, Science 210:537 (1980); L. Olsson et al, Proc. Natl. Acad. Sci. USA 77:5429; J. W. Pickering et al, J. Immunol. 129:406 (1982)). Unfortunately, these hybrid cells exhibited poor growth in vitro, low levels of antibody expression, instability of antibody expression, and a poor ability to be cloned by limiting dilution.
Consequently, diverse and cumbersome approaches have been used to produce human monoclonal antibodies. These include “humanizing” mouse antibodies by creating hybrid murine/hybrid immunoglobulin genes and generating antibodies in transgenic mice that bear human immunoglobulin gene loci. However, these methods are only able to produce antibodies that have been generated in mice by the murine immune system. They do not allow the isolation, production, and use of the naturally-occurring antibodies, the immunological memory that the human immune system produces in response to infections and other antigen exposures. The ability to make monoclonal antibodies directly from human B-lymphocytes is therefore needed and would be of considerable value.
Recently, there has been progress in generating human monoclonal antibodies by generating hybridomas using the SP2/0 cell line as a fusion partner. The SP2/0 cell line is an immortal murine myeloma cell line (a malignant B-lineage cell) that expresses an endogenous murine telomerase gene. U.S. Patent Application Publication No. 20030224490 discloses the genetic modification of the SP2/0 cell line to ectopically express interleukin-6 (IL-6) and human telomerase catalytic subunit (hTERT).
However, progress in making fully human monoclonal antibodies has been hampered by the absence of human myelomas suitable for use as fusion partners with the desirable attributes of mouse myeloma cells such as stability, and high antibody production. The use of Epstein-Barr virus (EBV) has proved to be quite efficient for human lymphocyte immortalization (Kozbor D, and Roder J., J. Immunology 1981; 127:1275; Casual O, Science 1986; 234:476), but has certain limitations such as low antibody secretion rate, poor clonogenicity of antibody-secreting lines and frequent loss of antibody expression.
Immortalized human B cells have been employed for monoclonal antibody production. This approach involve the steps of: (a) isolation of peripheral blood lymphocytes enriched in B cells; (b) transformation of the B cells with EBV-viruses or fusion with immortalized human lymphoblastoid cells, and (c) massive screening for the B cell transformants or hybridomas exhibiting the desired antigen-binding specificity. B cell transformation itself is an inefficient process yielding at best 0.1-10% stable transformants. Thus most B cells with the desired specificity are lost in the pool used for subsequent selection process. Whereas researchers have attempted to enrich the population of B cells expressing the desired immunoglobulin by in vitro immunization/activation with the antigen of interest, the activation is again inefficient in the sense that non-specific B cells also proliferate during this process. The identification of specific antibody producing B cells thus largely depends on the final stage of screening, during which tens and thousands of transformed B cell clones are tested for their abilities to bind the antigen. This approach is time consuming and labor intensive.
The production of high-affinity antibodies is dependent on B cells expressing antibodies that have undergone the process of somatic hypermutation, which is the result of a complex set of events that mostly occur within germinal centers (GC). A post-germinal center B cell is a cell that that has undergone somatic hypermutation of its immunoglobulin genes. After completing the germinal center maturation response, B cells can become either memory B-cells, which circulate in the blood and form the foundation of a future immune response against the original antigen, or plasma cells, which home to the bone marrow, terminally differentiate, and secrete immunoglobulins. The development of memory B cells and plasma cells takes place in germinal centers of lymphoid follicles where antigen-driven lymphocytes undergo somatic hypermutation and affinity selection, presumably under the influence of helper T cells.
Typically, to generate hybridomas that secrete human antibodies, human peripheral blood mononuclear cell populations (PBMCs) are fused with a fusion partner cells because human splenic mononuclear cells, which contain ˜40% B cells, are not readily available. PBMCs are readily accessible by routine phlebotomy, but contain only about 5% B-cells (Klein et al., 1997 Blood 89: 1288; Dessain et al., 2004 J. Immunol. Methods 291: 109; Tian et al., 2007 Mol Immunol 44: 2173). However, only about 15% of the B-cells available in peripheral blood express class-switched, post-(GC antibodies (Klein et al., 1997 Blood 89: 1288; Tian et al., 2007 Mol Immunol 44: 2173). Accordingly, fusions with unselected PBMCs commonly yield hybridomas that express IgM antibodies with germline sequences, which are not as desirable as class switched, post-GC antibodies. A disadvantage of prior art methods is the high background of IgM secreting hybridomas. The present invention serves to address the low yield and success rate in generating desirable IgG class switched, post GC antibodies.
Botulism is a life-threatening, flaccid paralysis caused by a neurotoxin produced by the anaerobic bacterium Clostridium botulinum. Botulinum neurotoxin poisoning (botulism) arises in a number of contexts including, but not limited to, food poisoning (food borne botulism), infected wounds (wound botulism), and “infant botulism” from ingestion of spores and production of toxin in the intestine of infants. Botulism is a paralytic disease that typically begins with cranial nerve involvement and progresses caudally to involve the extremities. In acute cases, botulism can prove fatal.
Botulinum neurotoxin (BoNT) is found in nature as seven antigenically distinguishable proteins (serotypes A, B, Cl, D, E, F, and G). Botulinum neurotoxin acts at neuromuscular junctions. In addition BoNT has been designated as a category A select bioterrorism agent by the United States Government because of its extreme lethality and its availability from environmental sources (Arnon et al., 2001 JAMA 285:1059-70; Greenfield and Bronze, 2003 Drug Discov. Today 8:881-8; Marks, 2004 Anesthesiol. Clin. North America 22:509-32). An inhaled lethal dose of BoNT for a 70 kg person is less than 1 microgram; 1 gram contains enough BoNT to kill one million people (Arnon et al., 2001 JAMA 285:1059-70). Thus, devastatingly lethal amounts of BoNT could easily be transported and distributed in secret. Because of the requirement for immediate and prolonged ICU support for exposure victims, a limited civilian exposure could easily overwhelm the intensive care unit capability of a typical American city (NIAID, 2002b).
The chief countermeasures for BoNT exposure have historically been the botulinum toxoid vaccine and therapeutic antibodies. The existing vaccine is an inactivated pentavalent toxoid that induces a potent neutralizing antibody response (Amon et al., 2001 JAMA 285:1059-70; Gelzleichter et al. 1999 J. Appl. Toxicol. Suppl. 1:S35-8; Siegel, 1998 Immunol. Res. 17:239-51). However, it has not been recommended for use in the general population because the naturally occurring disease is rare and widespread vaccination would render vaccinees resistant to BoNT, which may be required for medical indications such as blepharospasm, dystonia and torticollis (Bell et al., 2000 Pharmacotherapy 20:1079-91). Use of the toxoid vaccine following BoNT exposure is of no value because it is slow to induce a neutralizing antibody response (Amon et al., 2001 JAMA 285:1059-70).
The effectiveness of therapeutic antibody treatments for BoNT exposure is well established. BoNT-neutralizing immunoglobulin (BoNT-Ig) given prior to BoNT exposure can prevent or eliminate complications (Arnon et al., 2001 JAMA 285:1059-70; Gelzleichter et al. 1999 J. Appl. Toxicol. Suppl. 1:S35-8; Siegel, 1998 Immunol. Res. 17:239-51). BoNT-Ig given after exposure can prevent progression of symptoms, although it cannot reverse synaptic injury that has already occurred. However, the effectiveness of the currently-available BoNT-neutralizing antibodies is limited. Thus, there is a need for additional therapeutic antibodies for BoNT. The present invention satisfies this need.